Consistent with a critical role for altered Nav1.1 levels and PV cell–dependent activity in the pathogenesis of network and cognitive dysfunction of hAPP mice, reversing Nav1.1 reductions in hAPPJ20 mice by Nav1.1-BAC transgene expression restored PV cell–dependent gamma oscillations and inhibitory synaptic currents and reduced premature mortality, network hypersynchrony, and memory deficits in these mice. Thus, Nav1.1 depletion in PV cells may contribute to network dysfunction and cognitive deficits in hAPP mice and possibly in AD. Improving the function of PV cells and gamma activity may be of therapeutic benefit in AD and other brain disorders associated with altered network activity and cognitive impairments.

In support of the latter possibility, exploratory behavior was associated with increased gamma activity and fewer spikes in hAPPJ20 mice (Figure S1C). Interestingly, only hAPPJ20 mice with increased gamma activity during exploration had reductions in spikes (Figure 1G). Thus, gamma activity, but not exploratory behavior per se, modulates epileptiform discharges in these mice.

To determine if intrinsic properties of fast-spiking PV cells are altered in hAPPJ20 mice, we crossed hAPPJ20 mice with bacterial artificial chromosome (BAC) transgenic mice expressing enhanced GFP (eGFP) directed by GAD67 regulatory sequences (GAD67eGFP) (Chattopadhyaya et al., 2004) to produce GAD67eGFP mice without or with hAPP expression (NTG/GAD67eGFP and hAPPJ20/GAD67eGFP, respectively). eGFP-labeled fast-spiking GABAergic interneurons and nonlabeled pyramidal cells were assessed by patch-clamp recordings. Fast-spiking interneurons, but not pyramidal cells, had more depolarized resting membrane potentials in hAPPJ20/GAD67eGFP mice than in NTG/GAD67eGFP mice (Figure 2D and Table S1). Action potential properties were examined using current steps of increasing amplitude (800 ms, 20 pA steps). Although the pattern and frequency of action potentials across different stimulus intensities (0–320 pA above threshold) did not differ between genotypes (Figures 2E and S2A–C), fast-spiking interneurons had smaller action-potential amplitudes in hAPPJ20/GAD67eGFP mice than in NTG/GAD67eGFP mice both at resting membrane potentials and when resting membrane potentials were held constant at −70 mV (Figure 2F, G and Table S1). No differences in action potential amplitudes were found in layer II/III pyramidal cells (Figure 2G).

Reduced VGSC Levels in PV Cells of hAPPJ20 Mice and in AD Brains

Because action potential–driven synaptic activity depends on VGSCs (Catterall et al., 2010; Meisler and Kearney, 2005) (Figure S2D–F), we measured the levels of the four main VGSC subunits expressed in the central nervous system (Nav1.1, Nav1.2, Nav1.3, and Nav1.6) (Meisler and Kearney, 2005). hAPPJ20 mice had reduced levels of Nav1.1 and Nav1.6, but not Nav1.2 and Nav1.3, in the parietal cortex (Figure 3A). We also analyzed Nav1.1, Nav1.2, and Nav1.6 levels in the inferior parietal cortex of patients with AD and nondemented controls. In the AD cases, Nav1.1 levels were significantly depleted, Nav1.6 levels showed a clear trend toward depletion, and Nav1.2 levels were unchanged (Figure 3B).

VGSCs show distinct patterns of expression across neuronal populations (Lorincz and Nusser, 2008; Meisler and Kearney, 2005; Ogiwara et al., 2007), and the effects of reduced VGSC expression on network activity depend on the type of neuron affected. In agreement with previous reports (Lorincz and Nusser, 2008; Meisler and Kearney, 2005; Ogiwara et al., 2007), Nav1.1 mRNA was highly expressed by a small subset of neurons in the hippocampus and parietal cortex (Figures 3C and S3A). Double-labeling for Nav1.1 mRNA and PV showed near complete co-location of these gene products (Figures 3C and S3A). Nav1.6 mRNA was highly expressed by the majority of hippocampal neurons and a small subset in the parietal cortex (Figure S3B). Double-labeling for Nav1.6 mRNA and PV revealed near complete co-location in the parietal cortex (Figure S3B). Consistent with these results and the expression of eGFP in PV cells in line G42 (Chattopadhyaya et al., 2004), Nav1.1 and Nav1.6 were expressed by green fluorescent cells in the parietal cortex of GAD67eGFP mice (Figure 3D, E). Thus, Nav1.1 and Nav1.6 expression in the parietal cortex is restricted primarily to PV cells. Similar results were found in primary cell cultures (Figure S3C).

To ensure that these behavioral effects in hAPPJ20 mice were not due to motor dysfunction, we increased trial frequency to enhance learning. Under these conditions, we observed no abnormalities in habituation and dishabituation in phenytoin- and vehicle-treated hAPPJ20 mice (Figure 4F), suggesting that their increased exploratory behavior in our original paradigm was related to cognitive impairment rather than abnormal motor function. Retesting of fully habituated phenytoin-treated hAPPJ20 mice 10 days later again revealed prominent dishabituation (Figure 4F), suggesting enhanced forgetting. These results are consistent with other data indicating that aberrant network activity contributes to cognitive deficits in hAPP mice (Palop et al., 2007; Roberson et al., 2011; Roberson et al., 2007).

To determine if Nav1.1 alterations in hAPPJ20 mice contribute to cognitive decline, we assessed spatial learning and memory of mice in the Morris water maze. Compared with NTG controls, hAPPJ20 mice were severely impaired in the hidden platform component (days 1–5), but not the visible platform component (s1 and s2) of the test (Figure 7A), indicating specific spatial learning deficits. Increasing Nav1.1 levels by Nav1.1-BAC expression reduced these spatial learning impairments in hAPPJ20 mice and did not affect the performance of NTG mice (Figure 7A). In the probe trial (platform removed), only hAPPJ20 mice failed to cross the original platform location more often than equivalent locations in non-target quadrants (Figure 7B). hAPPJ20/Nav1.1 mice performed at control levels, indicating improved spatial learning and/or memory retention.

Context-dependent habituation and dishabituation to a novel environment were assessed in the open field. hAPPJ20/Nav1.1 mice habituated faster and more completely than hAPPJ20 mice (Figure 7C; trials 1–4), suggesting improved contextual learning. After four trials of testing, hAPPJ20/Nav1.1 mice, but not hAPPJ20 mice, were completely habituated and indistinguishable in their activity from NTG and Nav1.1 mice. Mice were retested in the same environment 5 and 15 days later to assess contextual memory. NTG and Nav1.1 mice had persistently low levels of activity at 5 and 15 days (Figure 7C), indicating normal contextual memory. Compared with these groups, hAPPJ20 mice showed increased activity at 5 days and a marked increase in activity at 15 days (Figure 7C), indicating profound dishabituation (forgetting). The activity of hAPPJ20/Nav1.1 mice was indistinguishable from that of controls at 5 days and intermediate between the activities of controls and hAPPJ20 mice at 15 days. Only hAPPJ20 mice had impaired memory retention (Figure 7D). Additional training revealed again that hAPPJ20/Nav1.1 mice habituated better than hAPPJ20 mice (Figure 7C; trials 7–10). After 10 trials, all genotypes were fully habituated and indistinguishable from each other. Thus, deficits in contextual learning and memory of hAPPJ20 mice depend on time and experience. Overall, our data show that increasing Nav1.1 levels in hAPPJ20 mice enhances their capacity for learning and memory.

Potential Clinical and Therapeutic Implications

Cortical and hippocampal hyperactivity of neuronal networks is an early event in AD pathogenesis and is associated with early amyloid deposition in nondemented humans with or without mild cognitive impairment (MCI) (Sperling et al., 2009). Impaired inhibition has been suggested as a potential mechanism of network hyperactivity (Busche et al., 2008; Palop et al., 2007; Palop and Mucke, 2010; Sperling et al., 2009). Our findings support this notion and suggest that behavioral interventions or pharmacological manipulations that increase gamma activity and/or reduce network hyperactivity have beneficial effects on cognitive functions in the presence of pathologically elevated Aβ levels. They also suggest that pharmacological interventions that reduce gamma activity (e.g., antiepileptic drugs affecting sodium channel function) could impair cognitive function in patients with AD or related disorders. In support of this observation, phenytoin increases AD risk in nondemented elderly people (Carter et al., 2007) and causes acute cognitive decline in Down’s syndrome patients with AD (Tsiouris et al., 2002). It needs to be determined whether these mechanisms of cognitive dysfunction are playing critical roles in all or only a subset of AD patients. Mouse and human data suggest that early-onset FAD patients with seizures might be particularly affected by these alterations.

In Situ Hybridization

Tissue preparation and in situ hybridization were performed as described (Palop et al., 2007). Antisense and sense RNA digoxigenin-labeled probes were generated from EST clones (Open Byosystems) (NCBI#: BE944238 for Nav1.1 and AI839069 for Nav1.6). Digoxigenin-labeled probes were detected with the HNPP fluorescent detection set (Roche), followed by standard fluorescence immunohistochemistry with polyclonal anti-PV (Swant) or anti-GFP (Molecular Probes) antibodies. Nav1.1- and PV-positive neurons were quantified in two sections, 100 µm apart. In all, 551 neurons positive for PV and/or Nav1.1 were identified and analyzed. The proportion of PV-positive cells expressing Nav1.1 mRNA (PV+/Nav1.1+) and the relative Nav1.1 mRNA signal intensity in PV cells were calculated for each genotype.

Slice Electrophysiology

sIPSCs were recorded at the reversal potential of ionotropic glutamate receptors (0 mV); sEPSCs were recorded at the reversal potential of GABAA receptors (−65 mV); mIPSCs and mEPSCs were recorded in the presence of 0.5 µM tetrodotoxin and 50 µM CdCl2. Signals were acquired with Axoclamp 2A, MultiClamp 700B, or Axopatch 200A amplifiers (Molecular Devices). Voltage- and current-clamp recordings were analyzed in Igor Pro (Wavemetrics) with custom-written procedures. For detection of synaptic events in voltage-clamp recordings, events were detected as deflections exceeding 5–7 pA above baseline mean. From each recording, 500–1000 consecutive events were sampled.

To analyze the intrinsic properties of neurons recorded in current-clamp mode, the input resistance was estimated at the resting membrane potential in response to 500-ms hyperpolarizing current steps of 20–40 pA. The membrane time constant and capacitance were estimated by fitting single exponentials to initial phases of these voltage responses. For analysis of action potential properties, neurons were recorded at resting or at −70 mV membrane potentials and depolarized with 800-ms current steps in 20-pA increments. See extended experimental procedures.

EEG Recordings

EEG activity in freely moving mice was recorded with the Harmonie 5.0b software (Stellate). Sharp-wave discharges were automatically detected by the Gotman spike detectors (Harmonie, Stellate). Spectral analysis was analyzed using a custom-written procedure running under IGOR Pro v6.12A. The gamma frequency band (gamma activity) represents the average of the spectral values in the 20–80-Hz range (Barth and Mody, 2011). Low, intermediate and high intensity of gamma activities were defined as minutes with values <30%, 30–60%, >60% of the total amplitude of the gamma activity. See extended experimental procedures.

Drug Treatments

For acute treatments, riluzole was dissolved at 2 mg/ml in 50% polyethylene glycol 400 in distilled water and injected IP at 20 mg/kg, and phenytoin was dissolved at 10 mg/ml in phosphate-buffered saline and injected IP at 100 mg/kg. For chronic treatment, phenytoin was dissolved daily in the drinking water at 0.15–0.75 mg/ml to reach final doses of 25, 50, 70, and 85 mg/kg/day.

Morris Water Maze

Mice were trained to locate the hidden platform over 5 consecutive days (two sessions of two trials per day, 4 h apart). Four hours after the last training session, the platform was removed, and a 60-sec probe trial was performed. Two days later, mice were trained to locate the visible platform over two sessions of two trials each. Performance was monitored with an EthoVision video tracking system (Noldus Information Technology). See extended experimental procedures.

Open Field Behavior

Exploratory locomotor activity was measured in an open field (automated Flex-Field/Open Field Photobeam Activity System). Mice were placed in one of four identical clear plastic chambers (40 × 40 × 30 cm) for 5 min (Figure 7) or 15 min (Figure 4). Total movements in the open field were reported. See extended experimental procedures.

Statistical analysis

Statistical analyses were performed with SPSS 10.0, STATA 11.2, or Prism 5.0. Experimenters were blinded with respect to genotype and treatment of mice and to diagnosis of human cases. See extended experimental procedures.

Supplementary Material

01

02

ACKNOWLEDGEMENTS

This work was supported by a Stephen D. Bechtel, Jr. Foundation Young Investigator Award to J.J.P.; National Institutes of Health Grants AG022074, AG011385 and NS065780 to L.M., NS002808, NS030549, AG5131, and AG18440 to E.M.; the Philippe Foundation Award to L.V.; the Coelho Endowment to I.M.; Pew and McKnight Foundations to A.C.K.; Epilepsy Foundation Postdoctoral Fellowship to E.O.M.; and facilities grants from Stephen D. Bechtel, Jr. and the National Center for Research Resources. We thank A. Escayg for the Nav1.1-BAC transgenic mice; J. Noebles and A. Gittis for helpful comments; G.Q. Yu, X. Wang, W. Guo, E. Pham, K. Bummer, I. Lo and D.H. Kim for excellent technical support; and G. Howard and S. Ordway for editorial review.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

SUPPLEMENTAL DATA

The supplemental material includes five supplemental figures, one supplemental table, and extended experimental procedures.